The progression of a proliferating eukaryotic cell through the cell-cycle checkpoints is controlled by an array of regulatory proteins that guarantee that mitosis occurs at the appropriate time. Protein phosphorylation is the most common post-translational modification that regulates processes inside the cells, and a large number of cell cycle transitions are regulated by, in addition to protein-protein interactions, the phosphorylation states of various proteins. In particular, the execution of various stages of the cell-cycle is generally believed to be under the control of a large number of mutually antagonistic kinases and phosphatases. A paradigm for these controls are the cyclin dependent kinases (CDKs), whose activity is required for the triggering of mitosis in eukaryotic cells (for reviews, see Hunt (1989) Curr. Opin. Cell Biol. 1:268-274; Lewin (1990) Cell 61:743-752; and Nurse (1990) Nature 344:503-508). During cell-cycle progression, CDKs appear to trigger a cascade of downstream mitotic phenomena such as metaphase alignment of chromosomes, segregation of sister chromatids in anaphase, and cleavage furrow formation.
The CDKs are subject to multiple levels of control. These proteins are positively regulated by association with cyclins (Evans et al. (1983) Cell 33:389-396; Swenson et al. (1986) Cell 47:861-870; Xiong et al. (1991) Cell 65:691-699; Matsushime et al. (1991) Cell 66:701-713; Koff et al. (1991) Cell 66:1217-1228; Lew et al. (1991) Cell 66:1197-1206) and activating phosphorylation by the cdk activating kinase (CAK) (Solomon et al. (1992) Mol Biol. Cell 3:13-27). Negative regulation of the cyclin/cdk(s) is achieved independently by at least two different mechanisms: binding of the inhibitory subunits (p21, p16, p15, p27 and p18) (c.f., Xiong et al. (1993) Nature 366,701-704; Harper et al. (1993) Cell 75:805-816; El-Deiry et al. (1993) Cell 75:817-825; Gu et al. (1993) Nature 366:707-710; Serrano et al. (1993) Nature 366:704-707; Hannon et al. (1994) Nature 371:257-261; Polyak et al. (1994) Cell 78:59-66; Toyoshima et al. (1994) Cell 78:67-74; Guan et al. (1994) Genes and Dev. 8:2939-2950) and by phosphorylation of conservative threonine and tyrosine residues, usually at positions 14 and 15 in cdk(s) (Gould et al. (1989) Nature 342:81-86; Krek et al. (1991) EMBO J. 10:3331-3341; Gu et al. (1992) EMBO J. 11:3995-4005; and Meyerson et al. (1992) EMBO J. 11:2909-2917).
The phosphorylation of CDC2 on Tyr-15 and Thr-14, two residues located in the putative ATP binding site of the kinase, negatively regulates kinase activity. This inhibitory phosphorylation of CDC2 is mediated at least in part by the wee1 and mik1 tyrosine kinases (Russel et al. (1987) Cell 49:559-567; Lundgren et al. (1991) Cell 64:1111-1122; Featherstone et al. (1991) Nature 349:808-811; and Parker et al. (1992) PNAS 89:2917-2921). These kinases act as mitotic inhibitors, over-expression of which causes cells to arrest in the G2 phase of the cell-cycle. By contrast, loss of function of wee1 causes a modest advancement of mitosis, whereas loss of both wee1 and mik1 function causes grossly premature mitosis, uncoupled from all checkpoints that normally restrain cell division (Lundgren et al. (1991) Cell 64:1111-1122).
A stimulatory phosphatase, known as cdc25, is responsible for Tyr-15 and Thr-14 dephosphorylation and serves as a rate-limiting mitotic activator. (Dunphy et al. (1991) Cell 67:189-196; Lee et al. (1992) Mol. Biol. Cell. 3:73-84; Millar et al. (1991) EMBO J. 10:4301-4309; and Russell et al. (1986) Cell 45:145-153). In humans, there are three known cdc25-related genes which share approximately 40-50% amino-acid identity (Sadhu et al. (1990) PNAS 87:5139-5143; Galaktionov and Beach (1991) Cell 67:1181-1194; and Nagata et al. (1991) New Biol. 3:959-968). Human cdc25 genes were recently found to function at G1 and/or S-phase of the cell cycle (Jinno et al. (1994) EMBO J. 13:1549-1556) in addition to the previously identified G.sub.2 or M-phase functions (Galaktionov and Beach, D. ibid.; Millar, et al. (1991) PNAS 88:10500-10504).
The c-myc protooncogene belongs to a family of related genes implicated in the control of normal cell proliferation and the induction of neoplasia. See, for example, Bishop, J. M. (1983) Annu. Rev. Biochem. 52, 301-354; Albrecht et al. (1992) Biochim. Biophys. Acta 1114, 129-146; Alitalo et al. (1987) Biochim. Biophys. Acta 907, 1-32; and Bornkamm, G. W. and Lenoir, G. M. In Advances in Viral Oncology, Vol 7. Klein, G. Ed.,173-206. Raven Press, N.Y. (1987). The first identified member of the family, v-myc, was found as a transforming gene of the MC29 and MHz avian retroviruses. A cellular homolog was later identified and isolated (Vennstrom et al. (1982) J Virol 42, 773-779). C-myc expression is associated with mitogenic activation in many cell types (Spencer et al. (1991) Adv. Cancer. Res. 56, 1-48; and Marcu et al. (1992) Annu. Rev. Biochem. 61, 809-860). Oncogenic activation of the gene in non-virally infected cells results from constitutive over-expression caused often by gene amplification, and has not been generally associated with mutation in the protein coding sequence (Lusher et al. (1990) Genes Dev. 4, 2025-2035; and Penn et al. (1990) Semin. Cancer Biol. 1, 69-80). More recently, c-myc has been implicated in the induction of apoptosis or programmed cell death (Askew et al. (1991) Oncogene 6, 1915-1922; and Evan et al. (1992) Cell 69, 119-128). Myc-induced apoptosis is enhanced by growth factor deprivation and the oncogenic-apoptotic switch is sensitive to the level of mitogenic stimulation.
The c-Myc protein (Myc) contains several structural motifs characteristic of sequence-specific transcription factors (Stone et al. (1987) Mol. Cell. Biol. 7, 1697-1709; Freytag et al. (1990) Cell Growth Differ. 1, 339-343; and Penn et al. (1990) Mol. Cell. Biol. 10, 4961-4966). The biological activity of Myc depends on the integrity of an N-terminal transactivation domain and a carboxy-terminal basic-helix-loop-helix-leucine zipper (b-HLH-LZ) domain (Kato et al. (1990) Mol. Cell. Biol. 10, 5914-5920; Blackwell et al. (1990) Science 250, 1149-1151; and Blackwood et al. (1991) Science 251, 1211-1217). The latter promotes association with Max, an essential partner, creating a sequence-specific DNA binding complex that recognizes a core hexanucleotide motif (CA(C/T)GTG). Myc alone binds DNA poorly and in vivo exist primarily in association with Max. Both Myc-Max and Max-Max dimers co-exist in equilibrium and bind to the same sequence motif in vitro. However, Myc overexpression activates, whereas Max overexpression represses, a CACGTG-driven reporter gene (Kretzner et al. Nature 359, 426429).
The function of Myc in oncogenesis and the induction of apoptosis is inhibited by mutations that inactivate transcription activation and leucine zipper domains. Complementary Myc and Max mutants, while defective in binding to the wild type partners, restore Myc transforming activity when coexpressed in cells, showing that Myc/Max interaction is essential for the Myc transforming activity (Amati et al. (1993) Cell 72, 233-245). Myc activity in cell cycle progression and apoptosis also requires the presence of both intact transactivation and Max binding domains. Thus, Myc displays the properties of a transcriptional activator which requires dimerization with Max to bind DNA. Following mitogenic stimulation of quiescent fibroblast cells the intracellular level of Myc is induced within 3-5 hours and then decreases to a constant intermediate level, whereas the level of Max is growth factor independent. Thus, the abundance of Myc appears to be the rate limiting determinant of Myc/Max activity (Kretzner et al. Nature 359, 426-429; and Ayer et al. (1993) Cell 72, 211-222.).
Few transcriptional targets of c-myc have been identified (Packham et al. (1995) Biochim. Biophys. Acta 1242: 11-28). One, ornithine decarboxylase (ODC), is an essential enzyme involved in the synthesis of polyamines. ODC displays certain oncogenic properties, suggesting that it might be an important c-myc target. Recently, it has been shown that induction of Myc in growth factor depleted cells causes rapid activation of the cyclin E/cdk2 kinase without alteration in the abundance of cdk2 or cyclin E (Steiner et al. (1995) EMBO J. 14:4814-4826).